It is well known that many therapeutic proteins (e.g., biologic drugs) have immunogenic potential, and administration of therapeutic proteins to a patient sometimes results in the production of antibodies against the therapeutic protein. Such anti-drug antibodies (ADA) may reduce the effectiveness of the therapeutic protein, for example they may bind to or/and neutralize the therapeutic protein, resulting in changes of drug pharmacokinetics or pharmacodynamics that alters drug efficacy. ADA may cause serious side effects, including allergic reactions, cross-reactivity against endogenous proteins, and complement activation. A life-threatening deficiency syndrome can result if ADA cross-reacts with and neutralizes a critical endogenous protein.
To screen for immunogenic activity of biologic drugs, assays for antibodies specific for potential therapeutic proteins, or components thereof, are often used during clinical drug development. Drug interference is regarded as one of the toughest challenges in such immunogenicity testing. For a drug with a long half-life and/or one administered at a high dose or a repeated dose, such as an antibody-based therapy, the ADA usually complexes with the drug, typically making the ADA unavailable for detection. In this situation, acid dissociation is often employed in an attempt to break up the ADA/drug immune complex so as to make the ADA available for detection. When a drug is present at high levels in the blood, it has proven extremely difficult to detect the ADA, even with acid dissociation, and it is often impossible to do so in a reproducible fashion.
Screening test subjects for the production of ADA is an important step in ensuring the safety and efficacy of many therapeutic proteins, and immunogenicity assessment of many therapeutic biologics (drugs comprising proteins) is required by regulatory agencies as part of the pre-clinical and clinical phases of drug development, as well as in the post-market phase to ensure their safety. See, e.g., US Food and Drug Administration (FDA) Draft Guidance for Industry: Assay Development for immunogenicity testing of therapeutic proteins, December 2009.
A variety of assay formats have been used with success to detect anti-drug antibodies, including ELISA (direct, indirect and bridging), radioimmunoassays, electrochemiluminescence, and surface plasmon resonance. The development of such assays, however, is often complicated by interference caused by the presence of the drug.
There is, thus, a need in the art for methods and kits for more accurately and reproducibly detecting anti-drug antibodies in samples (such as human blood fluids) that contain high levels of drug. The present invention fulfills this need, and provides other related advantages.